Recent cDN A microarray studies performed in our laboratory have indicated the presence of two dominant subtypes of metastatic melanoma, with potentially different clinical outcomes. cDNA microarray analyses of moles, primary and metastatic melanomas have identified a large number of genes whose expression level can be used to distinguish between the known landmarks in melanoma progression, including genes operating in the NF-kappaB and WNT-2 signaling pathways. In this application, we propose to extend these observations: Aim 1: To validate the prognostic significance of the molecular subtypes of metastatic melanoma identified by gene expression profiling in a large cohort of melanoma patients. We will confirm the prognostic impact of gene expression signatures using cDNA microarray analysis on a larger, prospectively identified cohort of metastatic melanoma patients. Aim 2: To use systemic, plasmid-based delivery of siRNAs, ribozymes, and cDNAs to characterize the functional roles of four genes identified from our array studies on the metastatic progression of melanoma in mice. We will characterize the functional importance of four genes in the NF-kappaB and WNT-2 signaling pathways on the metastatic progression of melanoma. We will use siRNAs and ribozymes targeting NCOA3, PHIP, and WNT2, and the WIF1 cDNA to validate the importance of each of these genes in the metastatic progression of melanoma. Aim 3: To examine expression levels of two candidate melanoma progression genes in a large tissue bank of human melanomas, and to correlate the expression of these genes with melanoma outcome. We will assess whether the protein products of NCOA3 and WNT2 provide new molecular markers to identify human melanoma patients at high risk for metastasis by performing immunohistochemical analysis of routine sections and tissue arrays, using an extensive bank of primary human melanomas. These studies should help validate the role of genes identified by expression profiling in the progression of melanoma.